Assessing Bioactivity-Exposure Profiles of Fr it and .... Wetmore_Food...Assessing Bioactivity-Exposure Profiles of Fr it and Vegetable J icesof Fruit and Vegetable Juices Barbara

Assessing Bioactivity-Exposure Profiles of Fr it and Vegetable J icesof Fruit and Vegetable Juices

Barbara A Wetmore Ph DBarbara A. Wetmore, [email protected]

July 28, 2016

Food Safety Specialty Section Webinar | July 28, 2016

The views expressed in this presentation are those of the author and do not necessarily reflect the views or policies of the U S EPAand do not necessarily reflect the views or policies of the U.S. EPA.


Providing Context to HTS Toxicity DataToxCast and Tox21 screening have generated millions of data points assessing

biological activities induced by synthetic chemicals.

Do these activities reflectadverse effect, or merely, yperturbations that couldoccur due to normal,everyday exposures?

Context is an extremely important yet often overlooked component to


Context is an extremely important yet often overlooked component to the interpretation of HTS data.

BioMAP

ToxCast on a Healthy Diet

BioMAPAssay Systems


BioMAP® Technology Platform

BioMAPAssay Systems

ReferenceProfile Database

Predictive Informatics Toolsy y

Human primary cells Disease-models

30+ systems

Biomarker responses to drugs are stored in the

database

Specialized informatics tools are used to predict

clinical outcomes


Human Biology Integrated into a Robust, Scalable Platform

BioMAP Diversity 8 Panel

3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT

BioMAP System

Venular endothelial cells

Venular endothelial cells

Peripheral blood mononuclear cells +

Endothelial cells

Peripheral blood mononuclear cells + Endothelial cells

Bronchial epithelial cells

Coronary artery smooth muscle

cellsFibroblasts Keratinocytes +

Fibroblasts

IL-1+ TNF-+ IFN-

IL-4+Histamine TLR4 TCR IL-1+ TNF-+

IFN- IL-1+ TNF-+

IFN-

IL-1+ TNF-+ IFN-+ EGF +

bFGF + PDGF-BB

IL-1+ TNF-+ IFN-+ TGF-

Primary Human Cell Types

Stimuli

13 7 11 10 11 14 12 9

Acute Inflammation E-selectin, IL-8E-selectin, IL-1, IL-8, TNF-, PGE2 IL-8 IL-1 IL-8, IL-6, SAA IL-8 IL-1α

Chronic Inflammation

VCAM-1, ICAM-1, MCP-1, MIG

VCAM-1, Eotaxin-3, MCP-1 VCAM-1, MCP-1 MCP-1, E-selectin,

MIG IP-10, MIG, HLA-

DRMCP-1, VCAM-1,MIG, HLA-DR

VCAM-1, IP-10, MIG

MCP-1, ICAM-1, IP-10

# of Endpoints

pes

Immune Response HLA-DR CD40, M-CSF

CD38, CD40, CD69, PBMC Cytotox., T cell Proliferation

HLA-DR M-CSF M-CSF

Tissue Remodeling uPAR, MMP-1, PAI-1, TGFb1, SRB tPA uPA

uPAR,

Collagen III, EGFR, MMP-1,

PAI-1, Fibroblast Proliferation SRB

MMP-9, SRB, TIMP-2, uPA,

TGFb1

Endp

oint

Typ

SRB, tPA, uPA Proliferation, SRB, TIMP-1

TGFb1

Vascular BiologyTM, TF, uPAR, EC Proliferation, SRB,

Vis

VEGFRII, uPAR, P-selectin, SRB Tissue Factor, SRB SRB

TM, TF, LDLR, SMC Proliferation,

SRB

Vascular Biology, Cardiovascular

Disease Chronic

Asthma, Allergy, Oncology,

Cardiovascular Disease, Chronic

Inflammation

Autoimmune Disease, Chronic

Inflammation

COPD, Respiratory,

Vascular Biology, Cardiovascular Inflammation

Tissue Remodeling,

Fibrosis Wound

Skin Biology,Psoriasis, Disease / Tissue Relevance


Disease, Chronic Inflammation Vascular Biology Inflammation,

Infectious DiseaseInflammation,

Immune Biology Epithelial Biology Inflammation, Restenosis

Fibrosis, Wound Healing Dermatitis

Hierarchical Clustering of Juice BioMAP Bioactivities


DiscoveRx BioMAP® Profile of Sweet PotatoesNon-cytotoxic doses

Key activities of Sweet Potatoes in Predictive Tox panel (continued): Tissue remodeling activities: Inhibition of uPAR (3C 4H); Increased MMP-1 (HDF3CGF) Tissue remodeling activities: Inhibition of uPAR (3C, 4H); Increased MMP 1 (HDF3CGF) Other effects: Increased EGFR (HDF3CGF) Top BioSeek Reference Database match: INCB-018424 SVM Mechanism Model Prediction: JAK Inhibitor


SVM Mechanism Model Prediction: JAK Inhibitor

Similarity Search Result for Sweet Potatoes15-deoxy-delta12,14-Prostaglandin J2

Overlay of Sweet Potatoes and 15‐deoxy‐delta12,14‐Prostaglandin J2P ’ l i 0 705 i i i d Pearson’s correlation, r = 0.705, common activities are annotated

15‐deoxy‐delta12,14‐Prostaglandin J2 is a PPARγ ligand that promotes adipocyte differentiation, induces heme oxidase (HO‐1) and is known to inhibit h NF B h


the NFκB pathway

DiscoveRx BioMAP® Profile of Strawberries

[Insert Annotated Profile here]

• Key activities of Strawberries:I fl ti l t d ti iti d d TNF VCAM 1 IP 10 MIG‐ Inflammation‐related activities: decreased sTNFa, VCAM‐1, IP‐10, MIG

‐ Immunomodulatory activities: decreased M‐CSF‐ Tissue remodeling activities: decreased PAI‐I; increased tPA


‐ Hemostasis‐related activities: increased TM, TF

Similarity Search Result for Strawberriestert-Butyl perbenzoate

Overlay of Strawberries and tert‐Butyl perbenzoatey y p Pearson’s correlation, r = 0.796, common activities are annotated tert‐Butyl perbenzoate is an organic peroxide used as a radical initiator to

induce polymerization


induce polymerization

Supervised Similarity Search Hits from BioMAP Library

BioMAP Similarity Search FindingsSupe v sed S y Se c s o o b y

Test Agent (%) Compound Hit (Conc) Compound Activities And/or Uses R Broccoli (0.46) Mitomycin C (370 ng/mL) alkylating agent, DNA cross-linker 0.806

Broccoli (0 46) Mancozeb (40 M) Broad-spectrum fungicide; anti-proliferative 0 774Broccoli (0.46) Mancozeb (40 M) effects in multiple cell types 0.774

Beets (0.46) Tannic acid (4.1 M) polyphenol, component in anti-allergen sprays 0.765

Kale (1.39) ZM449829 (3.3 M) JAK3 inhibitor tool compound binds to ATP site on enzyme 0.831

S P (4 17) ( ) Ruloxitinib, JAK 1/2 kinase inhibitor that 0 813Sweet Potatoes (4.17) INCB-018424 (10 M) Ruloxitinib, JAK 1/2 kinase inhibitor that improved survival in myelofibrosis patients 0.813

Green Beans (1.4) 15-deoxy-delta12,14-Prostaglandin J2 (3.3 M)

PPARγ ligand that promotes adipocyte differentiation, induces heme oxidase (HO-1)

and is known to inhibit the NFκB pathway 0.839

G B (0 46) T i id (4 1 M) l h l t i ti ll 0 804Green Beans (0.46) Tannic acid (4.1 M) polyphenol, component in anti-allergen sprays 0.804

Strawberries (0.46) tert-Butyl perbenzoate (40 M) organic peroxide used as a radical initiator to induce polymerization 0.796

Garlic (0.05) Oxibendazole (10 M) Antihelminthic 0.731

Grapes (0 46) tert Butyl perbenzoate (40 M) organic peroxide used as a radical initiator to 0 718Grapes (0.46) tert-Butyl perbenzoate (40 M) g pinduce polymerization 0.718

Apples (0.46) Tin(II) Chloride (30 M) HIF-1α inducer 0.705

Sweet Potatoes (1.4) 15-deoxy-delta12,14-Prostaglandin J2 (3.3 M)

PPARγ ligand that promotes adipocyte differentiation, induces heme oxidase (HO-1)

and is known to inhibit the NFκB pathway0.705


and is known to inhibit the NFκB pathway

Other activities of note: 6 juices with similarities to FICZ, an endogenous AhR ligand

Assessments Using Bioactivity Thresholds and Cytotoxicity Datay y y

Item Bioactivity Threshold Concentration (BTC), %

Lowest Concentration with Cytotoxicity

Bioactivity Margin( )

(# Hits)y y

(LCC), %g

Apples 0.02 (13) 4.17 245Bean Sprouts 0.05 (19) 12.50 245

Cabbage 0.02 (10) 4.17 245C f ( )Cauliflower 0.05 (15) 4.17 82

Kale 0.02 (12) 4.17 245Oranges 0.05 (9) 12.50 245Parsley 0.05 (21) 12.50 245Peppers 0 05 (11) 4 17 82Peppers 0.05 (11) 4.17 82Spinach 0.02 (26) 1.39 82

Sweet Potatoes 0.05 (23) 12.50 245Tomatoes 0.05 (12) 12.50 245

Concentration at which a minimum of 10% of the BioMAP assays

showed bioactivities

Ratio between BTC and LCC; used as an indicator of specificity of response


showed bioactivities of specificity of response

A Comment on Dosimetry…• Ongoing efforts to assess best strategy –g g gy

– to relate in vitro testing concentrations to an external anticipated exposure

• ToxCast chemicals and juices (i.e., phytochemical mixtures)

• Juices: Volume of juice yields per gram vegetable juiceJuices: Volume of juice yields per gram vegetable juice tracked– How to relate % v/v juice to a molar equivalent to allow

comparison to micromolar chemical concentrations? – Cmax comparison, assuming a certain degree of

bioavailability; rate and volume of distributionbioavailability; rate, and volume of distribution• Estimate of amount of juice contributing to bioactivity (adjust

for water content; nonbioactive content)


• … Stay tuned

Relating Bioactivities to ExposuresToxCast Phase I Chemicals; BioMAP Assays

Test Agent Bioactivity Threshold Conc. (M)

OED (mg/kg/

day)

mg/day, 75 kg male

Aggregated Max Detected

(mg/kg

Kg Produce consumed to achieve

produce) OEDAtrazine (31) 40.0 3.30 247.50 0.015 16500.00Azoxystrobin (46) 13.3 6.38 478.50 18.710 25.57Cyprodinil (40) 40.0 0.08 6.00 7.083 0.85Difenoconazole (37) 13 3 0 53 39 75 1 380 29 65Difenoconazole (37) 13.3 0.53 39.75 1.380 29.65Endosulfan (44) 13.3 0.05 3.75 0.790 4.75Fludioxonil (46) 13.3 0.18 13.50 19.200 0.70Hexythiazox (30) 13.3 0.40 30.00 0.850 35.29Pyraclostrobin (46) 1.5 0.29 21.75 14.163 1.54Pyraclostrobin (46) 1.5 0.29 21.75 14.163 1.54Simazine (39) 40.0 4.21 315.75 0.020 15787.50Zoxamide (13) 1.5 0.01 0.75 0.073 10.27

Pesticide residue data collated from USDA Pesticide Data Program (PDP)

Other chemicals assessed but with no detectable pesticide residues across the crops

Number in parentheses: number of crops analyzed for pesticide residues


tested: acetochlor, bendiocarb, benfluralin, fenitrothion, resmethrin, tefluthrin

Attagene and NCATS Profiling of Fruits and Veggies


NCATS Screening Data

Juice Hit Rate inNCATS Assays


Bioactivity‐Profile Comparisons – Attagene Platform

Fruit and Vegetable Juices ToxCast Phase I ChemicalsAssay Percent

Hit Rate

Number with 5-fold

increase

Max Responder Max Fold-Increase

over

Percent Hit Rate

Max Responder

Max Fold-Increase

overRate

(across 30

juices)

increase over Control

over Control

(across 309 chemicals)

over Control

NRF2_ARE_CIS 73 15 Spinach 14.9 52 Dichlorvos 16PPARg_TRANS 70 14 Bean Sprouts 20.9 46 Resmethrin 22

CRE_CIS 43 1 Garlic 5.2 16 Prallethrin 4.4AhR_CIS 43 8 Parsley 54.0 17 Tetraconazole 72

PXR_TRANS 37 0 Carrots 3.5 32 Fipronil 7.9Oct-MLP_CIS 37 1 Beets 5.9 27 Prallethrin 12

PPARa_TRANS 33 5 Bean Sprouts 9.7 3 Lactofen 11ERa_TRANS 33 3 Bean Sprouts 15.3 28 Pendimethalin 27_ pVDRE_CIS 33 0 Beets 3.4 42 Pyridaben 5.5AP-1_CIS 30 1 Cauliflower 5.7 16 Prochloraz 4.6


Nuclear Receptor (NR) Profiling / Cross‐Platform ComparisonsHierarchical Clustering

Vegetable Max Fold Induction over Control

Attagene Activities of Crucifers

AhR Nrf2/ARE PPAR‐ ER‐ PPAR‐Broccoli 11 11 9 3 5Cauliflower 23 21 21 3 10.3Cabbage 18 16 21 2 2Kale 10 11 9 2 4 5Kale 10 11 9 2 4.5

NR Activities of Note:• NRF2-ARE: antioxidant activities• PPAR-: BioMAP, ATG• AhR: BioMAP, NCATS, ATG

NR activity ≠ toxicity necessarily A broader exploration of the activities and potential


NR activity ≠ toxicity, necessarily. A broader exploration of the activities and potential roles/outcomes need to be explored for all chemical types.

Conclusions• Fruit and vegetable juices elicited a range of bioactivities acrossFruit and vegetable juices elicited a range of bioactivities across

the screening platforms assessed.• A suite of bioanalytic tools that utilize different informatics

approaches can prove useful in discerning:– Similarities to previously screened compounds;Potency (e g max fold change values)– Potency (e.g., max‐fold change values)

– Efficacy (e.g., curve‐fitting approaches)– Overall profiles (leverage whole dataset to inform re:Overall profiles (leverage whole dataset to inform re:specificity of activity; non‐specific and/or cytotoxic effects)

• A nuanced understanding of the overall dataset for any chemicalof interest is required prior to attributing an individual activitywith a particular mode of action or adverse outcome.

• Relating activities to an exposure metric using IVIVE or dosimetry


Relating activities to an exposure metric using IVIVE or dosimetryprovides an MOE context; useful as an assessment tool.

Acknowledgements

ScitoVation / SciMetrikaMichael DeVito (NTP)EPA/NTP/NCATS Collaborators

Mel Andersen Michael DeVito (NTP)Keith Houck (EPA – NCCT)Ruili Huang (NCATS)Richard Judson (EPA-NCCT)M tt M ti (EPA NCCT)

Mel AndersenMichael BlackBrian CholewaBriana FoleyB th P k Matt Martin (EPA-NCCT)

Russell Thomas (EPA-NCCT)Raymond Tice (NTP)Menghang Xia (NCATS)

Bethany ParksSalil Pendse

g g ( )

American Chemistry Council –Funding

DiscoveRxEllen Berg

American Chemistry Council Long Range Initiative

ILSI North America –Technical Committee on Food

d Ch i l S f t


and Chemical Safety

Documents

Assessing Bioactivity-Exposure Profiles of Fr it and .... Wetmore_Food...Assessing Bioactivity-Exposure Profiles of Fr it and Vegetable J icesof Fruit and Vegetable Juices Barbara